Recently, urinary trypsin inhibitor (UTI) has been widely investigated as an indicator of organism conditions, and various examinations have been carried out in the field of clinical medicine. UTI is known, for example, to appear in urine when an organism is exposed to inner or outer stress, such as inflammation, surgery, or the like, or is infected ("Clinical Significance of Urinary Trypsin Inhibitor" by Shiro KUWAJIMA et al., JAPANESE JOURNAL OF INFLAMMATION REVIEW ARTICLE, VOL9, NO. 3, MAY 1989).
While such usefulness has been pointed out, UTI has not been fully applied in the field of clinical medicine and the like due to the insufficient sensitiveness in the conventional UTI measuring method.
In other words, since UTI inhibits trypsin activity corresponding to its amount, the concentration or the activity of UTI is measured by determining the inhibitory level of the trypsin activity. As an example of this method, there is a method for measuring the enzyme activity by mixing a urine sample with a buffer solution, then mixing the resultant solution with an enzyme (trypsin) solution, and adding a substrate solution therein. Generally, this measurement employs calcium as a trypsin activator. The calcium is usually mixed in the buffer solution.
In this measurement, benzoyl-arginine-p-nitroanilide (BAPNA) is widely used as a substrate. When trypsin cleaves this substrate, colors come out. The trypsin activity is determined by measuring the colors with a spectrophotometer. However, since this synthetic substrate has poor solubility, a substrate solution having a concentration of 1.0 g/L or more is difficult to prepare. Consequently, the enzyme activity depends on the substrate concentration, and therefore it has been difficult to improve the trypsin activity. On the other hand, an extremely small amount of UTI inhibits the trypsin activity. As described above, the concentration or the activity of UTI is measured by determining the inhibitory level of the trypsin activity. Therefore, when the trypsin activity cannot be determined at high sensitivity, the concentration or the activity of UTI also cannot be measured at high sensitivity.
In order to solve this problem, BAPNA is dissolved in a polar organic solvent, and the solvent is then diluted with water to double the volume, thus preparing a substrate solution. However, the use of such organic solvents makes it difficult to apply the measuring method mentioned above in an autoanalyser. In addition, the organic solvent might damage plastic cells used in general autoanalysis devices. Furthermore, the organic solvent might inhibit the protease activity such as trypsin and the like. Moreover, even if preparing the substrate solution by using an organic solvent, the substrate might be crystallized and precipitated in long-term storage or cold storage. Consequently, in the conventional measuring method, when dissolving the slightly soluble substrate such as BAPNA or the like by using an organic solvent mentioned above, it has been necessary to prepare the substrate solution for every measurement and carry out the measurement directly after the preparation. In addition, even if preparing the substrate solution using the organic solvent, the UTI sensitivity was not sufficient.